The natural immune response against tumour antigens is relatively ineffective and various mechanisms allowing tumours to escape the anti-tumour immune response have been identified. Conventional vaccine approaches generate humoral responses which prove to be insufficient.
Strategies aimed at developing a cytotoxic response based on antigen-presenting cells (APCs), in particular based on dendritic cells, have been studied. The principle consists in specifically destroying the cancer cells of a patient by stimulating said patient's own immune defences.
Dendritic cells are antigen-presenting cells (APCs) that are very effective in generating cytotoxic effectors specific for tumour cells. They are capable of phagocytizing apoptotic cells or apoptotic bodies originating from tumour cells, and then of presenting the tumour antigens, in association with MHC class I and II molecules, to T lymphocytes. Thus, the dendritic cell is capable of initiating the proliferation and the generation of a clone of specific cytolytic T lymphocytes. At the end of this reaction, the killer lymphocytes thus differentiated leave the lymphoid compartment so as to circulate in the organism and bind in the tumour. Recognition of the antigens expressed by a tumour then induces a lytic signal and brings about the destruction of the tumour cells.
Several anti-cancer vaccine strategies targeting dendritic cells have been studied (Eymard J C, Bernard J, Bull Cancer. 2003, 90(8-9):734-43). Some are based on manipulating the dendritic cells in vitro, and others are based on stimulating the dendritic cells in vivo. In the first case, the dendritic cells are differentiated from blood cells taken from the patient: they are cultured and matured, “pulsed”, i.e. stimulated, ex vivo with tumour peptides, tumour lysates, apoptotic tumour cells, or heat shock proteins extracted from autologous tumours, and finally reinjected into the patient. In the second case, the stimulation of the dendritic cells is carried out after injection, into the patient, of peptides, proteins, irradiated tumour cells or else viruses containing the antigenic peptide targeting the dendritic cells. However, the cytotoxic response obtained is rarely accompanied by clinical efficacy. The activation of the dendritic cell “conditions” its ability to effectively activate the cytolytic T lymphocyte. The level of activation of dendritic cells appears to be a delicate point in this vaccine strategy.
The use of dendritic cells ex vivo for obtaining an anti-tumour vaccine raises a certain number of issues on: the state of maturation of the dendritic cells, the number of cells to be injected, the route, the site and the frequency of injection for producing dendritic cells capable of migrating to the secondary lymphoid organs and of inducing an effective cytotoxic T response (Banchereau J, Schuler-Thurner B, Palucka A K, Schuler G. [Dendritic cells as vectors for therapy] Cell. 2001, 10, 106(3): 271-4.).
The activation of dendritic cells in vivo is, for its part, limited by the weak immunogenic capacity of tumour antigens and the difficulty in activating dendritic cells at a sufficient level.
The use of red blood cells as carriers for transporting antigens, encapsulated in the red blood cells or bound to their surface, and delivered to the APCs has been envisaged in several publications. The triggered immune responses have been investigated in vitro and in vivo.
Hamidi et al. have recently described the encapsulation of BSA (bovine serum albumin) as a model of an antigen in human red blood cells (Hamidi M et al., Drug Deliv., 2007; 14(5):295-300 and Int J Pharm., 2007, 29, 338(1-2): 70-8). The authors suggested the use of red blood cells as a vector for the presentation of antigens to APCs of the reticuloendothelial system (RES). In another review published by Hamidi et al. (J. Control. Release, 2007, 118(2): 145-60), the authors indicated that a certain number of strategies have been studied to promote targeting of the RES, said targeting being promoted by ageing of the red blood cells, leading to their uptake for lysis. Other routes were mentioned, such as exposure of red blood cells to stabilizing agents, in particular crosslinking agents, coating of red blood cells with anti-RH antibodies, of IgG type to target the spleen or of IgM type to target the liver, heat shock, or exposure to oxidizing agents, enzymes or antibiotics.
A humoral immune response can be obtained in vivo after immunization with antigen-loaded red blood cells. The study carried out by Murray et al. made it possible to detect, in mice, IgG immunoglobulins after intravenous injection of murine red blood cells loaded with one of the following four antigens: KLH (Keyhole Limpet Haemocyanin), BSA (Bovine Serum Albumin), CTB (Cholera Toxin b Subunit) and ADA (Bovine Adenosine Deaminase). Detection of IgG1 and IgG3, which are predominant immunoglobulin isotypes during a Th2 response, and of IgG2, immunoglobulin isotype which is a marker for a Th1 response, would suggest the involvement of both types of immune responses, the humoral response and the cellular response (Murray A M et al., Vaccine., 2006 28, 24(35-36): 6129-39).
Another formulation of antigens used with red blood cells has been tested by Dominici et al. The Tat protein of the HIV-1 virus was anchored to the surface of mouse red blood cells by means of avidin/biotin coupling. The immunization of mice by intraperitoneal injection with this antigen formulation, internalized by dendritic cells, triggered a humorally-mediated immune response in vivo. Isotype characterization of the immunoglobulins detected indicates the induction of Th1 and Th2 responses. The anti-Tat cytotoxic activity was shown in vitro, by the conventional chromium-release technique, for the mice treated with red blood cells coupled to the antigens (Dominici S et al., Vaccine., 2003 16, 21(17-18): 2073-81).
A cellular response has been demonstrated in vitro by Corinti et al. with the same formulation. Phagocytosis of the red blood cells conjugated with Tat protein, by dendritic cells derived from human monocytes, was shown, as was the induction of CD4+ and CD8+ responses. Moreover, maturation of dendritic cells in the presence of interferon gamma promoted the type I immune response (Corinti S. et al., Leukoc. Biol. 2002, 71(4): 652-8).
Boberg et al. injected mice intraperitoneally with a vaccine constituted of peptides derived from the HIV-1 protease anchored at the surface of mouse red blood cells by means of the avidin/biotin system. They chemically modified red blood cells with the aim of promoting their recognition by APCs, but a weak immune reaction was obtained. They concluded that the small amount of antigens delivered, due to the limited loading of red blood cells with antigenic peptides and blood volume injected was not compensated for by the chemical modification of the carriers supposed to promote antigen recognition by APCs (Boberg A. et al., Infect. Agents Cancer., 2007, 182: 9).